1. Field of the Invention
This invention, in the field of immunology and medicine, provides compositions and methods for inducing enhanced antigen-specific immune responses, particularly those mediated by cytotoxic T lymphocytes (CTL), using chimeric or hybrid nucleic acid molecules that encode an endoplasmic reticulum chaperone polypeptide, e.g., calreticulin, and a polypeptide or peptide antigen. Naked DNA and self-replicating RNA replicon vaccines are provided.
2. Description of the Background Art
DNA vaccines have become an attractive approach for inducing antigen-specific immunotherapy. Forms of DNA vaccines include “naked” DNA, such as plasmid DNA (U.S. Pat. Nos. 5,580,859; 5,589,466; 5,703,055), viral DNA, and the like. Basically, a DNA molecule encoding a desired immunogenic protein or peptide is administered to an individual and the protein is generated in vivo. Use of “naked” DNA vaccines has the advantages of being safe because, e.g., the plasmid itself has low immunogenicity, it can be easily prepared with high purity and, compared to proteins or other biological reagents, it is highly stable. However, DNA vaccines have limited potency. Several strategies have been applied to increase the potency of DNA vaccines, including, e.g., targeting antigens for rapid intracellular degradation; directing antigens to antigen presenting cells (APCs) by fusion to ligands for APC receptors; fusing antigens to chemokines or to antigenic pathogenic sequences, co-injection with cytokines or co-stimulatory molecules or adjuvant compositions.
Cancer vaccines are an attractive approach for cancer treatment because they may have the potency to eradicate systemic tumor in multiple sites in the body and the specificity to discriminate between neoplastic and non-neoplastic cells (Pardoll (1998) Nature Med. 4:525-531). Anti-tumor effects of the immune system are mainly mediated by cellular immunity. The cell-mediated component of the immune system is equipped with multiple effector mechanisms capable of eradicating tumors, and most of these anti-tumor immune responses are regulated by T cells. Therefore, it is hoped that cancer vaccines, particularly as DNA vaccines, aimed at enhancing tumor-specific T cell responses will be developed to control tumors.
HPV oncogenic proteins, E6 and E7, are co-expressed in most cervical cancers associated with HPV and are important in the induction and maintenance of cellular transformation. Therefore, vaccines targeting E6 or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. HPV-16 E7, a well-characterized cytoplasmic/nuclear protein that is more conserved than E6 in HPV-associated cancer cells, has been exploited in a number of HPV vaccines.
Calreticulin (CRT), an abundant 46 kilodalton (kDa) protein located in the lumen of the cell's endoplasmic reticulum (ER), displays lectin activity and participates in the folding and assembly of nascent glycoproteins. See, e.g., Nash (1994) Mol. Cell. Biochem. 135:71-78; Hebert (1997) J. Cell Biol. 139:613-623; Vassilakos (1998) Biochemistry 37:3480-3490; Spiro (1996) J. Biol. Chem. 271:11588-11594. CRT associates with peptides transported into the ER by transporters that are associated with antigen processing, such as TAP-1 and TAP-2 (Spee (1997) Eur. J. Immunol. 27:2441-2449). CRT also forms complexes with peptides in vitro. Upon adminsitration to mice, these complexes, elicited peptide-specific CD8+ T cell responses (Basu (1999) J. Exp. Med. 189:797-802; Nair (1999) J. Immunol. 162:6426-6432). CRT purified from murine tumors elicited immunity specific for the tumor from which the CRT was taken, but not for an antigenically distinct tumor (Basu, supra). By pulsing mouse dendritic cells (DCs) in vitro with a CRT-peptide complex, the peptide was re-presented by MHC class I molecules on the DCs to stimulate a peptide-specific CTL response (Nair, supra).
CRT also has anti-angiogenic effects. CRT and a fragment comprising amino acid residues 1-180, which has been called “vasostatin,” are endothelial cell inhibitors that can suppress tumor growth (Pike (1999) Blood. 94:2461-2468). Tumor growth and metastasis depend on the existence of an adequate blood supply. As tumors grow larger, adequate blood supply to the tumor tissue is often ensured by new vessel formation, a process termed angiogenesis. (Folkman (1982) Ann. NY Acad. Sci. 401:212-27; Hanahan (1996) Cell. 86:353-364). Therapeutic agents that target and damage tumor vasculature can prevent or delay tumor growth and even promote regression or dormancy.
Self-replicating RNA vaccines (RNA replicons) have emerged as an important, more potent form of nucleic acid vaccines. RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (Xiong (1989) Science 243:1188-1191), Semliki Forest virus (Ying (1999) Nature Med. 5:823-827), or Venezuelan equine encephalitis virus (Pushko (1997) Virology 239:389-401) vectors. These vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA, which is then transcribed into RNA replicons in transfected cells or in vivo. (Berglund (1998) Nature Biotechnol. 16:562-565). Self-replicating RNA infects a diverse range of cell types and allows the expression of the antigen of interest at high levels (Huang (1996) Curr. Opin. Biotechnol. 7:531-535). Additionally, self-replicating RNA eventually causes lysis of transfected cells because viral replication is toxic to infected host cells (Frolov (1996) J. Virol. 70:1182-1190). These vectors therefore do not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for vaccine development targeting proteins that are potentially oncogenic, such as the HPV E6 and E7 proteins.
Chen (2000) Cancer Research 60:1035-1042 demonstrated that linkage of human papillomavirus type 16 (HPV-16) E7 antigen to Mycobacterium tuberculosis heat shock protein 70 (HSP70) leads to the enhancement of DNA vaccine potency. Other studies have demonstrated that immunization with heat shock protein (HSP) complexes isolated from tumor or virus-infected cells are able to induce potent anti-tumor (Janetzki (1998) J. Immunother. 21:269-276) or antiviral immunity (Heikema (1997) Immunol. Lett. 57:69-74). Immunogenic HSP-peptide complexes can also be reconstituted in vitro by mixing the peptides with HSPs (Ciupitu (1998) J. Exp. Med. 187:685-691). HSP-based protein vaccines can also be administered by fusing antigens to HSPs (Suzue (1996) J. Immunol. 156:873-879, HSP70 fusion protein elicited humoral and cellular immune responses to HIV-1 p24). These experiments demonstrate that 1) HSP-peptide complexes derived from tumor cells or virus-infected cells can stimulate tumor or virus-specific immunity; 2) the specificity of this immune response is caused by tumor-derived peptides that are bound to HSPs and not caused by the HSPs themselves; and 3) the immune response can be induced in mice with MHC either identical or different to the MHC of donor HSPs (Przepiorkia (1998) Mol. Med. Today 4:478-484; Srivastava (1998) Immunity 8:657-665). While these investigations have made HSPs more attractive for use in immunotherapy, the only HSP vaccines that have been tested thus far are in the form of protein-based vaccines or DNA-based vaccines.